The calcium-binding synaptotagmin family of vesicle proteins control vesicle fusion with the plasma membrane. The best studied family member, synaptotagmin I, is necessary for fast, synchronized neurotransmitter release from presynaptic terminals. While the roles of synaptotagmin I are being elucidated, the roles of the additional 14 members of the group are less clear. However, our preliminary studies support the notion that astrocytes, but not hippocampal pyramidal neurons, express synaptotagmin IV. Given that synaptotagmin IV knockout mice have demonstrated deficits in hippocampal-based learning and memory, the goal of project 3 is to test the hypothesis that synaptotagmin IV is essential for calcium-triggered exocytotic release of glutamate from astrocytes and that this regulated transmitter release pathway is critical for the control of synaptic transmission at the tripartite synapse. In the first aim we will test the hypothesis that astrocytic synaptotagrnin IV regulates glial transmitter release and the modulation of synapses in situ (Haydon, Ellis-Davies and Coulter). It is well established that trains of synaptic activity lead to long-term modifications of the synapse. We hypothesize that such activity similarly causes a long-term change in the astrocytic arm of the tripartite synapse. In collaboration with project 2, we will, in the second aim, ask whether synaptic activity regulates the synthesis of gliotransmitter release machinery within astrocytes in situ (Haydon, Ellis-Davies and Eberwine). The molecules studied in this experiment will be dependent on the initial results from project 2 where mRNAs resident within astrocytes and those specifically within glial processes will be identified. However, one candidate that we will study is synaptotagmin IV. To achieve this objective we will build on our recently developed ability to use electroporation to introduce mRNA into astrocytes in acutely isolated hippocampal slices. This aim is not feasible in a stand-alone project and will require close interactions as the outcomes of project 3 will dictate the directions taken here. By elucidating control functions of astrocytes over synaptic transmission this collaborative Conte Center project has the potential to change the way we view both short- and long-term regulation of the synapse.